We propose to elucidate the molecular mechanisms of arrestin-1 interactions with different functional forms of rhodopsin, which significantly contribute to exquisitely regulated and precisely timed function of photoreceptor cells, using the combination of novel cutting-edge biophysical methods and in vivo experiments. We will compare the shape of the complex of arrestin-1 with two forms of phospho-rhodopsin: rhodopsin carrying 3-4 phosphates, which are routinely formed in rod photoreceptors and appears to be perfectly harmless, and tight arrestin-1 complexes with hyper-phosphorylated rhodopsin that are cytotoxic in mice and Drosophila. We will explore the dynamics of free and rhodopsin-bound arrestin using high pressure EPR to elucidate the functional role of its flexibility. We will determine the biological role of arrestin-1 self-association in photoreceptors in vivo, which is currently unknown. Finally, we will test novel compensational approach to gene therapy of gain-of-function rhodopsin mutations using engineered enhanced arrestin-1 mutants capable of shutting off rhodopsin signaling independently of its phosphorylation. Proposed studies will significantly improve our understanding of photoreceptor physiology and advance our progress towards gene therapy of gain-of-function rhodopsin mutations. Due to high conservation of mechanics of GPCR regulation, mechanistic studies of arrestin-1 binding to rhodopsin will have broader implications, improving our understanding of the molecular basis of the function of other arrestin subtypes. Arrestin will be the first signaling protein studied by high-pressure EPR, which will reveal the functional role of its conformational flexibility. Protein-protein interactions, whch are often mediated by flexible or even unstructured elements, play key role in the regulation of cellular processes. Therefore, proposed studies will shed new light on many aspects of cell signaling. Our strategic goal is to gain sufficient understanding of protein-protein interactions that govern cell signaling to construct mutants with desired functional characteristics for research and therapeutic purposes.